This study examined the role of osteopontin (OPN), a phosphorylated secreted glycoprotein, in the promotion of trophoblastic cell migration, an early event in the embryo implantation process. Three human choriocarcinoma cell lines, namely JAR, BeWo, and JEG-3, were treated with variants of OPN differing in the extent of phosphorylation following sequential dephosphorylation with tartrate-resistant acid phosphatase (TRAP), and their migratory response was measured. The highly phosphorylated human milk form of OPN (OPN-1) strongly triggered migration in all three cell lines, whereas the less phosphorylated variants, OPN-2a and OPN-2b, failed to stimulate migration. JAR cell migration in response to OPN-1 was accompanied by a rapid rearrangement of actin filaments to the cellular membrane. Using broad spectrum protein kinase profiling, we identified p70 S6 kinase as a major signal transduction pathway activated by OPN-1 during the migratory response in JAR cells. Activation was blocked completely by rapamycin and LY294002, thus demonstrating that OPN-1-stimulated migration occurs through mTOR and PI3K pathways, respectively. Conversely, PD98059 did not affect the activation of p70 S6 kinase by OPN-1, therefore, this response does not involve the Ras/ MAPK signaling cascade. Together, these data show that the highly phosphorylated human OPN-1 can stimulate trophoblastic cell migration and provides evidence for the involvement of the PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response. Because both OPN and TRAP are expressed in the uterus during early pregnancy, it is conceivable that extracellular phosphatases such as TRAP may modify OPN charge state and thus modulate cell migration. © 2005 Wiley-Liss, Inc.